Endoderm Induction and Pancreatic Specification from ES*

描述(由申请人提供)： 胚胎干细胞在培养中产生多种细胞类型的能力为研究导致初级胚层(外胚层、中胚层和内胚层)发育的谱系承诺早期阶段的调控机制提供了前所未有的机会。此外，ES细胞为移植治疗多种疾病提供了一种新颖的、潜在的无限供应的细胞。为了充分实现ES细胞系统的基本生物学和治疗潜力，首先必须确定在该模型中调节谱系诱导和组织指定的机制。这项建议的目的是研究在小鼠和人类ES细胞分化培养中调节内胚层诱导和胰腺规范的机制。总体方法是将前三个目标中的每一个实验都集中在三个特定的发展阶段。在第一个目标中，我们将使用一种小鼠ES细胞系，该细胞系具有针对短臂基因的GFP基因和针对Foxa2(HNF3())基因的人CD4，以确定发育的最早阶段所需的信号通路，即建立原始的条纹样种群。诱导出可与原始条纹相媲美的群体是最终内胚层发育的第一步。第二个目标的重点是确定哪些因素调节原始条纹样细胞向内胚层和Pdx1+胰腺祖细胞的发育。第三个目标将确定Pdx1+祖细胞成熟为具有功能的胰岛素分泌细胞所需的条件。在第四个目标中，我们将把老鼠ES细胞研究的结果转化到人类系统中。类似于小鼠ES细胞分化的方法将被用来建立从人类ES细胞发育成原始条纹状细胞、内胚层、胰腺前体细胞和胰岛素分泌细胞所需的条件。对于这些实验，我们将使用以下人类ES细胞系：ES02、ES03、ES04、TE-03、TE-06、MI01、UC01、WA01、WA07、WA09、BG02和BG03。这些实验的结果将为ES细胞分化模型中内胚层诱导和胰腺指定的调节提供新的见解。

DESCRIPTION (provided by applicant): The ability to generate multiple cell types from embryonic stem (ES) cell in culture offers unprecedented opportunities to investigate the mechanisms that regulate the earliest stages of lineage commitment that lead to the development of the primary germ layers; ectoderm, mesoderm and endoderm. In addition, ES cells offer a novel and potentially unlimited supply of cells for transplantation for the treatment of a broad range of diseases. For the basic biological and therapeutic potential of the ES cell system to be fully realized, it is essential to first define the mechanisms that regulate lineage induction and tissue specification in this model. The goal of this proposal is to investigate the mechanisms that regulate endoderm induction and pancreatic specification in both mouse and human ES cell differentiation cultures. The overall approach is to focus the experiments in each of the first three aims on three specific stages of development. In the first aim, we will use a mouse ES cell line with the GFP cDNA targeted to the brachyury locus and human CD4 targeted to the Foxa2 (HNF3() locus to define the signaling pathways required for the earliest stage of development, the establishment of a primitive streak-like population. Induction of a population comparable to the primitive streak represents the first step in the development of definitive endoderm. The focus of the second aim is to determine what factors regulate the development of endoderm and Pdx1+ pancreatic progenitors from the primitive streak-like cells. The third aim will define the conditions necessary for the maturation of Pdx1+ progenitors to functional insulin secreting cells. In the fourth aim, we will translate the findings from the studies with mouse ES cells to the human system. Approaches comparable to those used for differentiation of the mouse ES cells will be used to establish conditions necessary for the development of primitive streak-like cells, endoderm, pancreatic progenitors and insulin-secreting cells from human ES cells. For these experiments we will use the following human ES cell lines: ES02, ES03, ES04, TE-03, TE-06, MI01, UC01, WA01, WA07, WA09, BG02 and BG03. The outcome of these experiments will provide new insights into the regulation of endoderm induction and pancreatic specification in the ES cell differentiation model